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Saturday, November 22, 2014

DNA Microarray – Types, Strategies and applications

DNA microarray is a hybridization technique performed in microscopic glass slide with surface modification, anchoring specific set of probes sequences complementary to the Target. Upon complementation with the target sequence fluorescence signal can be captured and state of the test sample can be identified.

DNA microarrays can be used to measure changes in expression levels, to detect single nucleotide polymorphisms (SNPs), or to genotype or targeted resequencing.

Principle of DNA Microarray

The principle behind microarray is the hybridization between two DNA strands. Complementary sequence of the DNA pair specifically by forming hydrogen bonds, the more number of complementary base pairs in a nucleotide sequence will have a tighter non-covalent bonding between the two strands.

DNA hybridization


After the hybridization of target DNA to the probes attached on the glass slide, slides are washed to remove any non-specifically bound sequences, only strongly paired strands will remain hybridized.

Fluorescently labelled target sequences that bind to a probe sequence generate a signal that depends on the hybridization conditions (such as temperature), and washing after hybridization. Total strength of the signal, from a spot (feature), depends upon the amount of target sample binding to the probes present on that spot. Microarrays use relative quantitation in which the intensity of a feature is compared to the intensity of the same feature under a different condition, and the identity of the feature is known by its position.


Preparing Glass Slide for Surface Modification

Glass slide used for surface modification microarray need to be clean, to remove any organic contaminats and dirt glass can be cleaned with Ethanol, Toulene or Etching with pirhana. Etching with pirhana (7:3 H2SO4:H2O2) is the better choice among  the other cleaning methods.  One need to be careful while while handling pirhana solution as it is a strong oxidizer.


Surface Modification Strategies

 Various Surface modification are developed for DNA microarray, few of the most commonly used ones are


  • Poly – L- Lysine,
  • 3-aminopropyltrimethoxysilane (APS),
  • 3-glycidoxypropyltrimethoxysilane (GPS) and 
  • Aldehyde or carboxylic acid.

Oligo attachment to the modified glass surface

  • Oligo attachment to PLL coated glass slide


Poly – L – Lysine hydrogen bonding with an oligonucleotide.


  • Oligo attachment to APS coated glass slide


3-aminopropyltrimethoxysilane (APS) hydrogen bonding with an oligonucleotide.

  • Oligo attachment to GPS coated glass slide



3-glycidoxypropyltrimethoxysilane (GPS) covalently bound to an amine-terminated oligonucleotide.

  • Oligo attachemnt to DAB Dendrimer modified glass slide

One-half of a DAB dendrimer hydrogen bonding with an oligonucleotide


Spotting Oligo Probes on the glass slide

  • Mechanical Spotting
  • Ink Jetting
Microarray Workflow




Microarray Detection / Capture



List of commercially availbale DNA Microarrays
  • ACLARA Bio Sciences 
  • GeneChipTM - Affymetrix 
  • LabChip - Caliper Technologies 
  • LabCD system - Gamera Bioscience 
  • Genetix Ltd 
  • Agilent 
  • Illumina 
  • Incyte Microarray Systems 
  • Nanogen 
  • Sequenom and 
  • GenoSensor - Vysis Inc. (Downers Grove, IL)
References

Technical Resources, Array It Corp
Technical Resources, Affymetrix
A Beginer's Guide to DNA microarrays

Saturday, November 15, 2014

Comparison of TAE and TBE Buffers used in Gel Electrophoreis - Advantages & Disadvantages

In molecular biology, TBE and TAE buffers are used for agarose and polyacrylamide gel electrophoresis.

TAE – Tris Acetic acid EDTA.
  • Common buffer used in labs for DNA agarose gel electrophoresis. 
  • TAE is used at pH 8.0. 
  • Generally TAE is Prepared as 50X Stock.





TBE – Tris  Boric Acid EDTA. 
  • Common buffer used in labs for DNA agarose gel electrophoresis. 
  • TBE is used at pH 8.3. 
  • Generally TBE is Prepared as 10X Stock. 



Comparison of TAE Buffer Vs TBE Buffer


Resolution : 

TAE is Good for separating Long DNA Fragments where as TBE is good for separating small / short DNA fragments.


Enzyme Compatibility (Downstream applications like Cloning) :

TAE is the preferred choice if the DNA is used for cloning or other downstream applications. TBE should not be used if the DNA is further used in cloning as the borate is strong inhibitor of many of the enzymes.

Buffering Capacity : 

TAE has low buffering capacity compared to TBE. TBE is stable and has high buffering capacity.

Migration of DNA:

In TAE buffer migration of Linear Double stranded DNA migrates faster. In TBE migration of DNA is slower when compared to the TAE. 

Cost:

When considering cost TAE buffer is inexpensive compared to TBE.


Thursday, November 13, 2014

DNA Topology - Enzymes and Modes of action on cccDNA

Twist Number:

Twist number is equal to the number of times one strand of covalently closed circular DNA (cccDNA) crosses in front of the other strand.

For right handed helices Tw > 0

Writhing Number:

Writhing number is equal to the number of times the double helix crosses over the long axis of the double helix in 3D space.

Types of Writhe

  • Interwound Writhe
  • Spiral Writhe

Interwound Writhe

For right handed interwound turn Wr < 0 and or left handed interwound turns Wr > 0

Spiral Writhe
For right handed interwound turn Wr > 0  and for left handed interwound turns Wr < 0

Linking Number = Twisting Number + Writhing Number

Topoisomerases:
  • Topoisomerases I
  • Topoisomerases II

Topoisomerases I

Type I Topoisomerases increases the linking number one step at a time.

Topoisomerase II 

Type II Topoisomerases increases the linking number two steps at a time.

DNA gyrase

Special type of topoisomerase II found in prokaryotes which decreases linking number by 2.
DNA gyrase requires ATP for activity.